Basic Virology Flashcards
Types of specimens for different infections
Resp –> swab (throat/nasal), aspirate (NPA, tracheal)
GI –> stool
Neuro –> CSF
Genital –> urethral or endocervical swab
Vesicular rash –> skin scraping/ swab, vesicular fluid
Most will need serum sample as well
Methods of Direct Detection in Viral Diagnosis: method, benefits, limitations
Immunofluorescence
- rapid point of care test (15-30 min)
- direct assay –> detect Ag using labelled Ab
- indirect assay –> serology (add Ab then labelled anti-IgG Ab)
- latex agglutination –> complex formation between Ag and Ab
- limitation: requires cell containing viral Ag – can’t use in serum, stool
Electron Microscopy
- low sensitivity (need 10^6 particles/ml to visualise)
- good for detection of virus in stool and skin (high titres)
- only detect morphology up to family level –> further use PCR or culture to differentiate
- benefits: rapid, high specificity
- limitations: technically demanding, low sensitivity
Histology/Cytology
- target disease tissue
- viral inclusion bodies: collections of replicating viral particles e.g. Owl’s eye in CMV
- limitations: low feasibility
Molecular Detection: method, benefits, limitations, conventional vs real-time
Applicable to solids and fluids
Not dependent on viability of virus
Nucleic acid hybridisation using direct detection of viral nucleic acid by probes
PCR most widely used
- amplify viral nucleic acid with subsequent analysis
- EXTREMELY SENSITIVE –> prone to contamination
- difficult to interpret +ve results –> latent viruses can give positives (but no disease)
- benefits: fast, specific, sensitive
- limitations: contamination, pre-requisite knowledge for making appropriate primer
Conventional PCR = detect and quantify products at the very end, using post-PCR analysis e.g. gel electrophoresis (technically demanding)
Real Time PCR = detect signals after every cycle and measure threshold cycle – quantify based on comparison to standard with known amount of DNA – high precision
(use probe with reporter and quencher dye –> binds to PCR products and cleaved by Taq DNA polymerase cycle –> releases dye which is proportional to amount of PCR products)
Viral culture: method, conventional vs rapid, benefits, limitations,
Inoculated and cultured cell lines (animal, tissue, embryonated egg)
- primary, semi-continuous and continuous cell cultures
Benefits: High sensitivity and specificity (growing live virus)
–> based on observation of cytopathic effect - changes in structure of cell due to viral invasion e.g. clumping, darkening, fusion of cells to form multinucleate cells, rounding into “grape-like” clusters
Conventional culture: 1-2 weeks (use in research)
Rapid culture: 2 days –> low speed centrifugation enhances infectivity of certain viruses e.g. CMV DEAFF (detection of early antigen fluorescent foci)
Limitations: conventional type time consuming, technically demanding
Serology: class specific vs function specific, methods, ELISA
Detection of Ab against Ag
Class specific
- Enzyme immunoassay, radioimmunoassay, IF
- IgM for acute infection (but not all), transient
- IgG for immunity (takes longer to increase)
- take one blood sample >1 week after illness onset
- positive or negative result
Function specific
- haemaglutination inhibition test, complement fixation test, neutralisation
concept: virus + patients serum would produce absence of cytopathic effect (neutralisation) or haemaglutination if there are virus neutralising Ab in serum - take 2 samples for acute and convalescent sera 10-14 days apart (to look for increase in Ab levels)
- no single diagnostic level
ELISA
- detect Ag –> wells coated with Ab -> add serum -> add enzyme-linked Ab (to Ag) -> wash -> add substrate specific to enzyme -> colour change = Ag +ve
- detect Ab –> beads coated with Ag -> add serum -> add enzyme linked anti-human Ab -> wash -> substrate -> colour change = +ve
Interpretation of Viral Diagnostics: sensitivities and specificities
Sensitivity
- serology: class>function
- viral detection: PCR > culture > others (PA > EM)
Specificity
- cross reaction e.g. CFT for HSV and VZV, IgM for Dengue and Japanese encephalitis
- non-specific reactions in unrelated infection
- timing of sampling - late first sample (already peaked), IgM too early, acute and convalescent sera too close together
Viral Morphologies: sizes, characteristic features of reoviridae, adenoviridae, caliciviridae, hepadnaviridae, orthomyxoviridae, paramyxoviridae, herpesviridae, papovavirdae, poxviridae
Reoviridae – 70-75 nm, “wheel” like
Adenoviridae – 70-75 nm, hexagonal with triangular faces
Caliciviridae – 30-35 nm, “Star of David”
Hepadnaviridae – 40-45 nm for complete virion, Dane particles/ Spherical form/ Tubular form
Orthomyxoviridae – 80-120 nm, peripheral fringe, pleomorphic
Paramyxoviridae – 90-300nm, shorter and less obvious peripheral fringe, pleomorphic, “herringbone-like” tubular structure
Herpesviridae – 95-105 nm, “fried egg”
Papovaviridae – 45-55 nm, skew arrangement
Poxviridae – 200-250 nm, dimorphic (mulberry and capsule), capsule with threadlike structures over it
Innate Immunity in Viral Infections: characteristics, mechanism of recognition, main 1st line immune response features, innate cells and their functions
Rapid, immediate, non-specific –> suppress viral replication early
PAMP e.g. dsRNA of nucleic acid is recognised by PRR (pattern recognition receptors) e.g. TLR on cell membrane or intracellularly –> activated cascade of events promoting innate immunity
- increase IFN-1 (main innate immunity in viral infections)
- released by plasmacytoid dendritic cells and macrophages
- effects: antiviral by blocking viral replication, immunomodulation of T/B/NK and cytotoxic T cells, prodromal symptoms (fever, malaise, fatigue)
- not virus specific but species specific and induces antiviral state in neighbouring non-infected cells too - inflammatory cytokines e.g. IL1, IL-6, TNF
- cellular recruitment, phagocytosis, initiate adaptive immune response - complement system
- acute phase proteins
Main cells in innate immunity:
- NK cells (cytotoxic, produces IFN-gamma to induce other antiviral responses), PDC (viral sensors, potent IFN-1 secretion), DC (Ag presenting, +ve adaptive), Macrophages (inflammation, high levels of IFN-1)
Adaptive Immunity in Viral Infections: characteristics, humoral response effects, cellular response effects
Develops over time (1-2 weeks), eliminates infection, provides memory (augment response), specific
Humoral response:
- stimulated B cells –> plasma cells secreting Ab
- effects: neutralisation (agglutinate virions, prevent attachment), opsonisation, ADCC (Ag-Ab-complement to lyse cells), interfere uncoating
Cellular response:
- Treg cells (helper: suppressor in 2:1) – either promote or inhibit other T and B cells
- cytotoxic T cells attack and lyse infected cells
Rmb MHC presentation of Ag in order for successful recognition by T cells
Clinical Applications of Immunity: immunisations and their features, examples of passive immunisation (3), other applications (2)
Active immunisation (vaccines)
- depend on active response from immune system
- takes a few weeks to develop
- lasts for years (have memory)
Passive immunisation
- enriched virus-specific Ab (pooled or specific Ab)
- no active response
- immediate protection but short lived (1-3mths)
- option for post-exposure prophylaxis
e.g. IVIG - pooled Ig from healthy donors with mixture of Ab against common infections
Specific Ig - high conc of specific Ig against certain virus from recovered donors e.g. Hep B IG, Zoster IG
Synthetic - using recombinant DNA tech e.g. monoclonal IgG against RSV for prevention in high-risk group
Other applications:
- recombinant interferons to augment immune response (less used nowadays)
- adjuvant in vaccines to augment response e.g. non-specific stimulation of TLR to produce higher levels of Ab
Route of entry of viruses and examples, stages of viral infection (5)
Skin - abrasions, inoculations, insect/ animal bite
Mucous membranes - conjunctiva, respiratory tract, GI tract, genital tract
Skin as barrier to microbes –> some degree of trauma allows virus to reach basal cells and start infection
— percutaneous infections e.g. HBV, HIV, Dengue start in cell types other than skin
Mucous membrane –> direct access to receptors on mucosal epithelial cells
– site of entry not necessarily ultimate site of infection e.g. rubella, VZV via pharynx and disseminate in bloodstream/ enterovirus via GI affects CNS/ skin
Stages of viral infection:
- entry –> regional LN for viral replication –> primary viraemia –> disseminate to target organs –> released from target organ causing secondary viraemia OR shedding of virus when amount exceeds threshold (transmission)
Patterns of Viral infection: incubation time, mechanism/ outcome, vaccination use, examples
Acute
- short incubation
- complete clearance and form memory
- vaccination useful
e. g. Hep A, Influenza, rubella
Latent
- have immune escape strategies
- establish latency which is not cleared by antivirals –> inactive, not replicating (dormant), non-infectious
- reactivate upon immunosuppression or stimulation
- vaccinations less useful
e. g. Herpesvirus family (HSV: neurons, VZV: dorsal root ganglia, EBV: B lymphocytes)
Chronic
- have immune escape strategies
- initially asymptomatic and may develop complications later
- continuous viral replication for years (active, infectious)
- only occurs in a proportion of infected subjects
e. g. HBV, HCV, HIV
Persistent = latent + chronic
Interactions between virus and host cells: pathology (4), autoimmunity and immunosuppression
Pathology:
- tropism (infect specific cells) e.g. HBV hepatotrophic
- cytopathic effects
- —- e.g. cell lysis - death and release of virions in non-enveloped virus (lytic infection) vs budding in enveloped virus (less damage to host)
- —- fusion of cells in RSV – multinucleated giant cells
- inclusion bodies (aggregation of viral proteins in nucleus or cytoplasm, altered staining) e.g. Owl’s eye in CMV
- immune mediated damage e.g. HBV specific T cells causing hepatocyte damage; IFN cause fever and malaise
Autoimmunity:
- molecular mimicry (post-infection or vaccination) e.g. GBS after influenza vaccine, post-infectious encephalitis after influenza
Immunosuppression:
- significant number of critical immune cells killed e.g. CD4 T cells in HIV
- alternation cytokine response in infected cells
- induction of Treg
- e.g. measles –> paradoxical immunosuppression for weeks increasing risk of secondary bacterial infection
